The long-term objective of this research project is to contribute to our understanding of how cell fates are specified during development. This course of study has broad health-related applications. For example, cancer results from inappropriate cell fate choices. In order to understand the abnormal development of cancer cells, we must first understand normal development. The free-living soil nematode, Caenorhabditis elegans, is an excellent model system in which to study cell fate. its amenability both to genetics and molecular biology, in addition to the fact that its complete cell lineage is known, make it an ideal choice for genetic, molecular, and cellular approaches to the analysis of development. The specific goal of this project is to identify and characterize genes and elements involved in aspects of vulval development that are not well understood. First, a reporter gene construct will be used as a molecular marker of vulval precursor cell fate to identify trans regulatory factors and cis regulatory elements required for its specific expression in the six vulval precursor cells. Trans-acting factors will be identified using an in vivo screen to isolate animals carrying mutations that cause aberrant expression of the reporter gene. Cis-acting elements will be identified by modifying the reporter gene and assessing the effect of that modification on reporter gene expression. These experiments will identify genes and elements required for vulval precursor cell identify. Second, genetic and molecular approaches will be used to identify and characterize the transcriptional targets of LIN-31, a transcription factor involved in the proper specification of vulval cell fates. These targets will identify gene products that re involved in the determination and/or expression of particular vulval cell fates. Specifically, a genetic screen for suppressors and enhancers of the lin-31 mutant phenotype will identify downstream regulators of vulval development, some of which may be LIN-31 targets. Molecular approaches to identify LIN-31 targets involve the determination of LIN-31 DNA binding sites using sequential selection of either partially degenerate oligonucleotides or genomic clones to an immobilized LIN-31 fusion protein. Finally, two approaches will be used to obtain mutations in these candidate LIN-31 target genes. One approach involves identifying existing mutations that map near the target genes and rescuing their mutant phenotypes by germline transformation. The second approach relies on a PCR-based screen to identify insertions of the Tc1 transposable element into candidate target genes. The research described in this proposal will generate the tools that will be used to dissect the process of cell fate determination during C. elegans vulval development. Knowledge of how cells choose their fates in this simple model system will shed light on the developmental processes of more complex organisms.